Open-channel block of Na+ channels by intracellular Mg2+

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Abstract
1. Macroscopic and single-channel currents through several types of cloned rat brain Na+ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na+ channels in chromaffin cells, intracellular Mg2+ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na+ channel (Pusch et al. 1989). 3. A sodium-channel mutant (‘cZ-2’) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg2+. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg2+ and a voltage-dependence of e-fold per 39 mV (in ≈125 mM [Na] i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg2+ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] i . This indicates that Mg2+ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 108 M−1 s−1 and 3.6 · 105 s−1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg2+ strongly depends on [Na] i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg2+ block at physiological levels of [Mg] i .